Hydrophobic cue-induced appressorium formation depends on MoSep1-mediated MoRgs7 phosphorylation and internalization in Magnaporthe oryzae

The rice blast fungus Magnaporthe oryzae forms specialized infectious structures called appressoria that breach host cells to initiate infection. Previous studies demonstrated that the regulator of G-protein signaling (RGS)-like protein MoRgs7 undergoes endocytosis upon fungal sensing of hydrophobic environmental cues to activate cAMP signaling required for appressorium formation. However, the mechanism by which MoRgs7 internalizes and its fate remains undetermined. We here show that MoSep1, a conserved protein kinase of Mitotic Exit Network (MEN), phosphorylates MoRgs7 to regulate its function. MoRgs7 phosphorylation determines its interaction with MoCrn1, a coronin-like actin-binding protein homolog that also modulates the internalization of MoRgs7. Importantly, the endocytic transport of MoRgs7 is critical for its GTPase-activating protein (GAP) function important in cAMP signaling. Together, our findings revealed a novel mechanism by which M. oryzae activates MoRgs7-mediated hydrophobic cue-sensing signal transduction involving protein phosphorylation and endocytic transport to govern appressorium formation and fungal pathogenicity.

Introduction Integrity (CWI) MAP kinase MoMkk1 indispensable in the development and virulence of M. oryzae [21]. Intriguingly, we found that MoSep1 also phosphorylates MoRgs7, modulating its binding affinity with the actin-binding Coronin-like protein MoCrn1, and that MoCrn1 is involved in the endocytosis of MoRgs7. Thus, MoSep1 is linked to Gα-cAMP signaling through MoSep1-dependent MoRgs7 phosphorylation and MoCrn1 binding. Our findings revealed a novel mechanism of MoRgs7-mediated hydrophobic cue signal transduction in the appressorium formation and pathogenesis of the blast fungus.

MoRgs7 is phosphorylated during appressoria formation
MoRgs7 is important in hydrophobic surface-induced appressorium formation in M. oryzae [20]. To investigate the underlying relationship between MoRgs7 and hydrophobic surface, we first examined whether MoRgs7 is phosphorylated by comparing conidial and appressorial stages using Mn 2+ -Phos-tag gel electrophoresis. We found that the band representing the phosphorylated-MoRgs7-GFP (P-MoRgs7) was apparent in the appressorial stage but not the conidial stage. The P-MoRgs7 band was absent in the presence of a phosphatase but more prominent in the presence of a phosphatase inhibitor (Fig 1A). To examine whether MoRgs7 phosphorylation is also responsive to hydrophilic cues, we compared proteins extracted from germinated conidia after 4 h of incubation on hydrophobic and hydrophilic surfaces. The result showed that MoRgs7-GFP is phosphorylated only when cultivated on the hydrophobic surface. This agrees with MoRgs7 subject to phosphorylation regulation during the early appressorial stage when recognizing hydrophobic surface cues (Fig 1B).

MoRgs7 is phosphorylated by MoSep1
To identify protein kinases that phosphorylate MoRgs7, we screened a Y2H cDNA library of M. oryzae using BD-MoRgs7, conducting by cloning full-length cDNAs of MoRgs7 into pGBKT7, as a bait vector and identified MoSep1 (MGG_04100) (S1 Text), a cell cycle-related kinase homolog, as a potential candidate. We further verified the interaction by Y2H and demonstrated that MoRgs7 and MoRgs7 Cterm interacts with both MoSep1 and MoSep1 STK (Fig  2A). To further examine the MoRgs7 and MoSep1 interaction, we generated two partial clones of MoSep1, MoSep1 STK , and MoSep1 BACK , and carried out co-IP and GST-pulldown assays. The assays showed that MoRgs7 interacts with both MoSep1 and MoSep1 STK but not MoSep1-BACK (S1A and S1B Fig).
To identify MoSep1-dependent phosphorylation sites, we compared LC-MS/MS analysis data between the wild-type (WT) Guy 11 and the ΔMosep1 mutant, and identified five differentiated serine phosphorylation sites, including S541, S544, S546, S547, and S550 (Figs 2B and S2). Each individual phosphorylation site was mutated to a negatively charged amino acid, aspartic acid, to mimic phosphorylation status, or to a non-phosphorylatable amino acid, alanine. After checking the phenotype and interaction relationship of each single mutant of MoRgs7, we found that every single mutation affects the MoSep1 and MoRgs7 interaction (S3 Fig). We then performed all five sites Serine (S) to Alanine (A) site-directed mutagenesis to mimic the phospho-dead mutant of MoRgs7 (MoRgs7 5A ), and Serine (S) to Aspartic acid (D) site-directed mutagenesis to mimic the phosphomimetic (MoRgs7 5D ) status. The mutant constructs were linked to either GFP or GST marker proteins. Phosphorylation assays with in vivo Mn 2+ -Phos-tag gel analysis and in vitro fluorescence detection in tube (FDIT) confirmed the phosphorylation relationship between MoSep1 and MoRgs7 depends on these serine phosphorylation sites (Fig 2C and 2D).

MoRgs7 phosphorylation is important for pathogenicity
To examine if MoRgs7 phosphorylation affects fungal pathogenicity, susceptible rice seedlings (CO-39) were sprayed with conidia of the wild-type Guy11, the ΔMorgs7 mutant, and the complemented ΔMorgs7/MoRGS7 strains, as well as the site-directed ΔMorgs7/MoRGS7 5A and ΔMorgs7/MoRGS7 5D mutant strains. Very few lesions were found in leaves infected with ΔMorgs7 and ΔMorgs7/MoRGS7 5A in comparison to ΔMorgs7/MoRGS7 5D and other strains (Fig 3A and 3B). Appressorial penetration and invasive hyphal growth assays indicated less than 25% appressorial penetration by the ΔMorgs7 and ΔMorgs7/MoRGS7 5A strains at 24 hpi MoRgs7-GFP proteins were extracted from transformants at conidia and appressoria stages, then treated with phosphatase and phosphatase inhibitors and shifted by Mn 2+ -Phos-tag SDS-PAGE and normal SDS-PAGE with the anti-GFP antibody. For total protein extraction, 1 mL lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40 [Sigma-Aldrich, I3021]) with 2 mM PMSF (Beyotime Biotechnology, ST506-2), proteinase inhibitor cocktail (Sigma-Aldrich, 11836170001) and deacetylation inhibitors (50 mM nicotinamide, 50 mM sodium butyrate, 5 mM Trichostatin A [Sigma-Aldrich, T1952]) were used to resuspend ground powder of appressoria and conidia. (B) Phosphorylation analysis of MoRgs7-GFP in Guy11 (the steps are similar to A). MoRgs7-GFP proteins were extracted from conidia of transformants which were allowed to germinate on hydrophobic and hydrophilic surfaces at 4 h-post inoculations.
We also compared the proportion of appressorium penetration in onion epidermal cells, and the results indicated that ΔMorgs7 and ΔMorgs7/MoRGS7 5A were defective in penetration when compared with Guy11 (S4A Fig). Further tests using various concentrations of glycerol to assess appressorial turgor pressure showed apparent differences. At 1 M of glycerol, the appressorial collapse rate was higher in ΔMorgs7 and ΔMorgs7/MoRGS7 5A strains than that in Guy11 and the ΔMorgs7/MoRGS7 5D strain. Moreover, the proportion of collapsed appressoria  [21]. aa, amino acid. Yeast two-hybrid analysis. MoRgs7 and its two segments MoRgs7 RGSDomain and MoRgs7 Cterm were co-introduced with MoSep1, MoSep1 BACK , and MoSep1 STK into the AH109 strain, respectively. The transformants were plated on SD-Leu-Trp and on SD-Leu-Trp-His-Ade for 5 days. (B) Phosphorylation sites were identified by comparing the wild type (Guy11) with the ΔMosep1 mutant expressing MoRgs7 in LC-MS-MS (Q-E) analysis. Pos., positive control. Neg., negative control. (C) In vitro phosphorylation analysis by the fluorescence detection in tube (FDIT) method. In the presence of 50 μm ATP, the purified proteins of GST-MoRgs7, GST-MoRgs7 5A , and His-MoSep1 were constructed for protein kinase reactions. The fluorescence signal at 590 nm (excited at 530 nm) was measured in a Cytation3 microplate reader (Biotek, Winooski, VT, USA). Error bars represent SD and asterisks represent significant differences (**p < 0.01). (D) Phosphorylation analysis of MoRgs7 in vivo. MoRgs7-GFP from corresponding transformants in appressorial stages was treated with a phosphatase (PE) and a phosphatase inhibitor (PI). Proteins were detected by the GFP antibody and shifted by Mn 2+ -Phos-tag SDS-PAGE and normal SDS-PAGE, respectively. in ΔMorgs7/MoRGS7 5A continuously increased at a faster rate than that in Guy11 and ΔMorgs7/MoRGS7 5D strains until glycerol reached 4M. These data suggested that MoRgs7 phosphorylation plays a role in maintaining turgor pressure. (S4B Fig).
The effect of MoRgs7 phosphorylation in appressorium formation evokes that the defect of RGS family proteins leads to abnormal perception of hydrophobic cues and could form two appressoria from a single conidium [19]. Examining the effect of MoSep1-dependent MoRgs7 phosphorylation on appressorial development showed that the percentage of conidia forming two appressoria was approximately 15% in the ΔMorgs7 and ΔMorgs7/MoRGS7 5A strains, while lower than 5% was recorded in ΔMorgs7/MoRGS7 5D (Fig 3D and 3E). These results suggested that MoSep1-dependent MoRgs7 phosphorylation is involved in appressoria induction.
It has been reported that MoSep1 is involved in Mitotic Exit Network (MEN) and Cell Wall Integrity (CWI) signaling that directly affects the pathogenicity of M. oryzae [21]. To examine if MoSep1-dependent MoRgs7 phosphorylation promotes MoSep1 function in MEN and CWI signaling, we expressed MoRgs7 5D -GFP constructs into ΔMosep1 and examined its vegetative growth, conidiation, and virulence. The results showed that continuous phosphorylation of MoRgs7 cannot restore the growth and virulence defect of ΔMosep1 (S5 Fig).

MoRgs7 phosphorylation is involved in MoMagA-mediated cAMP signaling and normal appressorial induction
MoRgs7 is one of the RGS family proteins that also plays an indispensable role in cAMP signaling in M. oryzae [19,20,22]. To examine if MoSep1-dependent phosphorylation affects the RGS function of MoRgs7, we assessed in vitro GTPase accelerating protein (GAP) activities of MoRgs7, MoRgs7 5A , and MoRgs7 5D . The result revealed that the activated MoRgs7 5D mutant exhibited the highest GAP activities, while the unphosphorylated MoRgs7 5A mutant showed the lowest GAP activities. This finding suggested that MoRgs7 phosphorylation is required for its GAP function ( Fig 4A).
The rate of GTPase activity directly affects intracellular cAMP levels. An examination revealed high intracellular cAMP levels in ΔMorgs7 and ΔMorgs7/MoRGS7 5A than that in Guy 11 and the ΔMorgs7/MoRGS7 5D complemented strain ( Fig 4B). MoRgs7 was previously found to interact only with Gα MoMagA [19,20,22]. MoMagA is one of the three Gα subunits (MoMagA, MoMagB, and MoMagC) and plays a major role in being part of Gα subunits that activates the downstream cAMP pathway [19,[23][24][25]. We generated BD-MoMagA G187S and MoMagA G187S -His constructs that mimic GTP-bound Gα [24] to examine if changes in GTPase accelerating protein activities involve affinity bindings between GTP-bound MoMagA and MoRgs7. Y2H, in vivo co-IP, and microscale thermophoresis (MST) assays all showed that MoMagA G187S bound with MoRgs7, with the highest affinity being with MoRgs7 5D (Figs 4C, 4D, S6A and S6B). In summary, MoSep1-dependent MoRgs7 phosphorylation directly enhances cAMP signaling by exhibiting GAP activities towards MoMagA in M. oryzae.

Phosphorylation is vital for MoRgs7 internalization
We have previously found that MoRgs7 couples with the actin-binding protein MoCrn1 to undergo endocytosis [20]. We then tested the hypothesis that MoSep1-dependent MoRgs7 phosphorylation also involves actin-dependent endocytosis. Using GFP fusion protein constructs, we found that MoRgs7 5A -GFP fluorescence was enhanced at the plasma membrane (PM), similar to MoRgs7-GFP in the ΔMosep1 mutant but in contrast to MoRgs7 5D -GFP, indicating that MoRgs7 internalization is dependent on MoSep1 (Fig 5A).
Previous studies also indicated that a coronin-like actin-binding protein, MoCrn1, directs MoRgs7 for endocytosis upon sensing hydrophobic cues [20]. To test if MoSep1-dependent MoRgs7 phosphorylation affects its binding affinity with MoCrn1, we assessed the interactions between various constructs (MoRgs7, MoRgs7 5A , and MoRgs7 5D ) and MoCrn1 by Y2H, in vivo co-IP, and MST assays (Figs 5B, 5C, S7A and S7B). The results demonstrated that the balance of MoRgs7 phosphorylation is critical for its binding with MoCrn1. The sustainable phosphomimetic MoRgs7 (MoRgs7 5D ) had a high affinity for MoCrn1, while the sustainable unphosphorylated MoRgs7 (MoRgs7 5A ) interacted weakly with MoCrn1. The above results demonstrated that MoSep1-dependent MoRgs7 phosphorylation enhances the bind of MoRgs7 to MoCrn1, which regulates MoRgs7 internalization.

Phosphorylation of MoRgs7 promotes its degradation via the ubiquitinproteasome pathway (UPP)
Previous studies suggested that MoRgs7 may undergo protein degradation following appressorium maturation and that the degradation is linked to UPP [26]. Monitoring the fluorescence signal with the same setting and of MoRgs7-GFP and MoRgs7 5A -GFP during appressorium maturation, we found that MoRgs7-GFP fluorescence disappears in approximately 80% of appressoria at 8 hpi, in contrast to MoRgs7 5A -GFP, whose fluorescence remained at the PM at the same time (Fig 6A and 6B). To further examine whether the disappearance of MoRgs7 involves UPP, we utilized the anti-ubiquitin (Ub) antibody to detect the ubiquitin chain of in vivo-purified proteins. After determining the sample loading of eluted proteins quantitatively by anti-GFP, MoRgs7 and MoRgs7 5D were more intensively stained with the anti-Ub antibody than MoRgs7 5A , suggesting that MoRgs7 degradation involves functions of UPP (Fig 6C).

Discussion
M. oryzae is the causal agent of rice blast, infecting its host by forming the appressorium that penetrates the host cell. The rice surface builds the primary barrier for host-pathogen interactions, and the characterization of rice surface cues, including hydrophobicity and hardness, is crucial for the early stages of appressorium formation [6]. It is believed that the pathogen surface receptor proteins such as GPCRs sense and internalize environmental cues to activate downstream effectors leading to large-scale gene expression reprogramming resulting in new development processes such as appressorium formation [27]. Receptor internalization undergoes a series of strict regulations, and, while still partly unclear, the regulation involves posttranslational modifications (PTMs), including the phosphorylation of intracellular residues that regulate GPCR activities [28]. Our previous studies revealed that the RGS-like domainand 7-TM domain containing MoRgs7 senses hydrophobic environmental cues and regulates cAMP signaling through MoCrn1-mediated endocytosis [20]. Here, we further demonstrated that MoRgs7 is phosphorylated by MoSep1 in response to hydrophobic surface cues to undergo the endocytic process and that MoSep1-dependent MoRgs7 phosphorylation and endocytosis are critical for the appressorial development and pathogenicity of M. oryzae.
RGS and RGS-like proteins are directly linked to the Gα-cAMP signaling pathway, which works as negative regulators to enhance intrinsic GTPase activities of GTP-bound Gα subunits, thereby inactivating G protein function [29,30]. Various studies explore the unique function of RGS proteins by focusing on their phosphorylation. For example, the S. cerevisiae RGS protein Sst2 is regulated by a phosphorylation feedback loop involving the MAP kinase Fus3 in response to pheromone stimulation [31]. In M. oryzae, the phosphorylation of RGS proteins also plays a significant role in fungal development. For example, MoRgs1 phosphorylation by the casein kinase 2 MoCk2 is required for appressorium formation and pathogenicity [32]. Our current studies provided compelling evidence to indicate that MoRgs7 is phosphorylated by the cell cycle-related kinase MoSep1 in response to hydrophobic surface cues, and the phosphorylation and endocytic transport links signaling transmission to fungal pathogenicity.
To establish and maintain a signal transmission balance of cell, cell membrane receptor proteins are redistributed from the PM to cytosol. This dynamic process is vital for GPCR-regulated development processes [33]. Previous studies focused on the process of GPCR trafficking as a way to understand their functional mechanisms. A. thaliana AtRGS1 endocytosis is regulated by AtWNK8-mediated phosphorylation that sustains environmental sugar signaling [14]. The endocytosis of yeast Ste2p requires the recruitment of Ste2p to preexisting clathrincoated pits (CCPs). This recruitment is regulated by receptor phosphorylation and subsequent ubiquitination [34]. In M. oryzae. we have previously reported that the actin-binding protein MoCrn1 directs MoRgs7 to endocytic pits/vesicles for GPCR internalization [20]. Here, we observed that MoCrn1 displays a higher affinity with phosphorylated MoRgs7, suggesting that MoSep1-dependent MoRgs7 phosphorylation is the key to MoCrn1-mediated MoRgs7 internalization. By revealing protein phosphorylation in GPCR endocytosis as a regulatory mechanism of GPCRs, our studies further revealed the complex and multitude of signaling transduction in M. oryzae.
MoSep1 functions as a Ser-Thr kinase necessary for septum formation and also links the MEN pathway to CWI signaling [21,35]. Our current studies revealing the additional role of MoSep1 indicate MoSep1 as a protein kinase of many different functions. Intriguingly, the represent the standard deviations, and asterisks denote statistical significance (**p < 0.01). (C) In vivo ubiquitination analysis of protein extracted from MoRgs7, MoRgs7 5A , and MoRgs7 5D transformants and purified with GFP-Sepharose.
https://doi.org/10.1371/journal.pgen.1010748.g006 phosphomimetic MoRgs7 suppressed the defect of appressorium formation in the ΔMorgs7 mutant but not the virulence defect of ΔMosep1, suggesting that MoSep1 mediated MoRgs7 phosphorylation is independent of its regulatory mechanisms in MEN and CWI pathways (S8A and S8B Fig).
In summary, we have identified MoRgs7 phosphorylation by MoSep1 as a novel functional mechanism of MoRgs7 in regulating hydrophobicity-induced cAMP signaling in M. oryzae. MoRgs7 contained a GPCR-like 7-TM motif in addition to its RGS-like domain and was identified to function in sensing hydrophobic environmental cues [8,20,36]. We have demonstrated that MoRgs7 phosphorylation by MoSep1 is required for signal transmission through MoCrn1-dependent endocytosis and the ubiquitin proteasome pathway. In addition, MoSep1-dependent MoRgs7 phosphorylation regulates intracellular cAMP levels for normal appressorium formation and pathogenicity of M. oryzae. Our studies promote a further understanding of pathogenesis mechanisms during rice blasts and benefit the discovery of novel disease management strategies (Fig 7).

Strains and cultural conditions
Guy11 was used as the wild-type strain of M. oryzae and cultured on a complete medium (CM). For vegetative growth, small mycelium blocks from 5-day-old colonies were transferred into fresh media and incubated at dark 28˚C. Mycelia were harvested from liquid CM medium grown for 2 days and used for DNA and protein extractions [37,38]. The wild-type and mutants were cultured on an SDC medium at 28˚C for 3 days in the dark and transferred to continuous illumination under fluorescent before being scraped for surface hyphae and conidia evaluation [26].

Protein extraction and Western blot analysis
For total protein extraction, strains were incubated in liquid CM media with shaking for 2 days and harvested. Mycelia were grounded into fine powder in liquid nitrogen and suspended in lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5mM EDTA, 0.5% NP-40, and 2 mM PMSF). The lysates were collected into 2.0 ml tubes in ice for 30 min and shaken every 10 min. Lysates were then centrifuged at 15,000 rpm for 10 min at 4˚C and supernatants were collected as total protein extracts [39]. For GFP-tagged protein detection, samples were analyzed with 12% SDS-PAGE gel and immunoblotted with anti-GFP antibodies (mouse, 1:5000, Abmart, 293967). Signals were detected by the ODYSSEY infrared imaging system (software Version 2.1). For ubiquitin chain detection, samples were analyzed using the anti-Ub antibody (mouse, 1:5000, ptmbiolabs, PTM-1107) [40].

Phosphorylation analysis
For in vitro analysis, GST-MoRgs7, GST-MoRgs7 5A , and His-MoSep1 were expressed in E. coli DE3 cells and purified [41]. We used the Pro-Q Diamond Phosphorylation gel stain (Thermo Fisher Scientific), a phosphor-protein gel-staining fluorescence dye in this assay. A kinase reaction buffer (100 mM phosphate-buffered saline, pH 7.5, 10 mM MgCl 2 , 1 mM ascorbic acid) was mixed with MoRgs7 and His-MoSep1, MoRgs7 5A , and His-MoSep1, respectively. The subsequent experiments were performed according to the previously described protocol [21].
For in vivo analysis, conidia were prepared from various transformants as described above and were filtered through three layers of lens paper before resuspending in sterile water (2 ×10 5 spores mL -1 ) [42]. For appressorium protein extraction, droplets (5 mL) of spore suspensions were placed on strips of onion epidermis, incubated under humid conditions at room temperature for 6 h, and onion epidermis grounded for protein extraction [43]. Protein extraction was the same as described above and phosphorylation analysis was performed as according to the protocol, phosphatase inhibitors (P0044, sigma) and alkaline phosphatase (P6774, sigma) [21].

Virulence test
Rice seedlings (Oryza sativa cv. CO39) were used for the pathogenicity test. Two-week-old rice seedlings were sprayed with 5 mL of conidia suspension and kept in darkness for 24 hours. Then rice seedlings were transferred into a transparent growth chamber following a 12 h/12 h light/dark cycle exposure schedule. After seven days, leaf disease severity was documented by photography. Lesions were quantitated by ImageJ [44].
3-week-old rice sheaths were collected and injected with conidia suspensions (2 × 10 5 spores/ml) with syringes. After 36 hours post-incubation at dark 28˚C, epidermal cells were observed by microscopy. This experiment was conducted with three biological repetitions and three replicates under the same experimental conditions [45].

LC-MS-MS analysis and GTPase activity assays
Fungal proteins were extracted as described above and separated by 10% SDS-PAGE gel electrophoresis. Phosphorylation site identification by LC-MS-MS was carried out as described previously [21].
His-MoRgs7, His-MoRgs7 5A , His-MoRgs7 5D , and His-MoMagA G187S were expressed in E. coli DE3 cells and purified. An ATPase/GTPase Activity Assay kit (MAK113; Sigma-Aldrich; Merck, Darmstadt, Germany) was used to assess the GTPase activity. The phosphate standards included in the kit were used to plot the standard curve. Reactions were incubated for 0 s, 1 min, 5 min, and 10 min at room temperature. Reagent buffer (200 ml) was added to each well, and incubation was carried out for an additional 30 mins at room temperature to terminate the enzyme reaction and generate the colorimetric end product. Absorbance at 620 nm was read and the change in absorbance values (DA620) was calculated by subtracting the control well (A620) from the sample well (A620). The concentration (μM) of free phosphate [Pi] was computed in the sample from the standard curve. The relevant experimental procedures were as described previously [32].

Intracellular cAMP level measurement
M. oryzae strains and transformants were cultured in liquid CM for 48 h and harvested. Mycelia were grounded into fine powders in liquid nitrogen and total cAMP levels were quantified by HPLC following previously established procedures [32].

Microscale thermophoresis assay
Bindings of MoCrn1 to MoRgs7, MoRgs7 5A , MoRgs7 5D , and MagA G187S to MoRgs7, MoRgs7 5A , and MoRgs7 5D were determined by MST using Monolith NT.115 (Nano Temper Technologies) according to the manufacture provided protocol. MST premium-coated capillaries (Monolith NT.115 MO-K005) were used to load the samples into the MST instrument at 25˚C using medium MST power. Laser on and off was set at 30 and 5 s, respectively. This experiment was repeated with three biological repetitions and three replicates under the same experimental conditions. Data were analyzed using Nano Temper Analysis software v. 1.2.101 (Nano Temper Technologies) [40]. . Samples were centrifuged at 3,600 rpm for 10 min, and the supernatants were transferred to new 1.5-mL tubes and stored at -70˚C. The GST-MoRgs7 supernatant was mixed with 30 mL of GSH Sepharose beads, incubated at 4˚C for 4 h, and then incubated with His-MoSep1, His-MoSep1 back , His-MoSep1 STK , and His supernatants at 4˚C for another 4 h. Finally, the beads were washed with buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM PMSF, 1% Triton X-100) 5 times and proteins eluted. Eluted proteins were analyzed by immunoblot (IB) with anti-His and anti-GST antibodies [46].

Appressorium formation and turgor pressure estimation
Conidia were harvested from 7-day-old SDC cultures and adjusted to 5×10 4 conidia ml -1 in sterile water. Droplets (30 μl) of conidial suspension were applied on coverslips (Fisher Scientific, St Louis, MO) and incubated under humid conditions at 28˚C prior to appressorial formation assessment [47].
The turgor pressure of mature appressorium was measured using an incipient cytorrhysis (cell collapse) assay. The 1.0-4.0 M glycerol solution was used to measure turgor pressure according to the previous description [21]. Collapsed appressoria were calculated based on data from at least 100 appressoria. This data had three replicates [21].

Statistical analysis
Results are presented as the mean ± standard deviation (SD) of at least three replicates. Significant differences between replicates were statistically evaluated using SDs and one-way ANOVA in SPSS 2.0. Results of two different treatments were statistically compared by ANOVA, followed by the F-test if the ANOVA result was significant at a p value of <0.05 or <0.01. Pathogenicity assay. The experimental procedures were conducted as the same as described for Fig 3. (B) Yeast two-hybrid analysis. MoSep1 was co-introduced with MoRgs7 and its each site-directed mutant into the AH109 strain, respectively. Transformants were plated on SD-Leu-Trp (as control) and SD-Leu-Trp-His-Ade (for further selection) for 5 days. (TIF) S4 Fig. MoSep1-dependent MoRgs7 phosphorylation is required for appressorium function. (A) The cytorrhysis assay was conducted by incubating conidia on the hydrophobic surface and treated with various concentrations of glycerol (1-4 M). Numbers of collapsed appressoria among 100 appressoria were recorded and the examination was repeated twice. Error bars represent SDs and asterisks represent significant differences (**p < 0.01). (B) Observation for penetration on the rice sheath. The penetration rate was counted 3 times. Error bars represent SDs and asterisks represent significant differences (*p < 0.05). MoMagA G187S was co-introduced with MoRgs7 and the site-directed mutagenesis mutants MoRgs7 5A and MoRgs7 5D into the AH109 strain. The transformants were plated on SD-Leu-Trp (as control), SD-His-Leu-Trp (for initial selection), and SD-Leu-Trp-His-Ade (for further selection) for 5 days. (B) Co-IP assay for the interaction between MoMa-gA G187S , MoRgs7 and its site-directed mutagenesis MoRgs7 5A and MoRgs7 5D . Co-expression of MoMagA G187S -RFP and MoRgs7-GFP, MoRgs7 5A -GFP and MoRgs7 5D -GFP in Guy11, respectively. Proteins were incubated with anti-RFP beads and detected by anti-GFP and anti-RFP antibodies. (TIF) S7 Fig. MoCrn1 interacts with MoRgs7, MoRgs7 5A , and MoRgs7 5D . (A) Yeast two-hybrid analysis. MoCrn1 was co-introduced with MoRgs7 and its site-directed mutagenesis MoRgs17 5A and MoRgs7 5D variants into AH109 strain, respectively. Transformants were plated on SD-Leu-Trp (as control), SD-His-Leu-Trp (for initial selection), and SD-Leu-Trp-His-Ade (for further selection) for 5 days. (B) Co-IP assay for the interaction between MoCrn1, MoRgs7 and its site-directed mutagenesis MoRgs7 5A and MoRgs7 5D . Co-expression of MoCrn1-RFP and MoRgs7-GFP, MoRgs7 5A -GFP and MoRgs7 5D -GFP in the wild-type strain Guy11, respectively. Proteins were incubated with anti-RFP beads and detected by anti-GFP and anti-RFP antibodies. (TIF) S8 Fig. MoSep1-dependent MoRgs7 phosphorylation is independent of CWI and MEN signaling in M. oryzae. (A) Hyphae of Guy11 and the ΔMosep1, ΔMosep1/MoSEP1, and ΔMo-sep1/MoRGS7 5D mutants expressing the H1-RFP construct were stained with CFW and examined by epifluorescence microscopy. Scale bar: 10 μm. (B) Hyphae of Guy11 and the ΔMosep1, ΔMosep1/MoSEP1, and ΔMosep1/MoRGS7 5D mutants were stained with CFW and examined by epifluorescence microscopy. Percentages shown in all images were calculated by the observation of 100 randomly-selected hyphae, and the observation was repeated twice. Scale bar: 10 μm. (TIF) S1 Text. Identification of MoRgs7 binding proteins. MoRgs7 was inserted into vector pGBKT7, and was were co-introduced with a yeast two-hybrid cDNA library contained various stages RNS of Magnaporthe oryzae into yeast AH109 strain. Then, AH109 strain was incubated on SD-Leu-Trp and SD-Leu-Trp-His-Ade for 5 days. The names of known protein filled in the brackets. The underlined protein is mentioned in this research. Among this table, MoMagA (MGG_01818) and MoCrn1 (MGG_06389) has been proved been interacted with MoRgs7 in previous studies (Li et al., 2019b;Zhang et al., 2011b). (DOCX)